Monoclonal gammopathy of undetermined significance (MGUS) is one of the most common pre-malignant disorders and affects approximately 3.5% of the population over 50 years of age. This grant application aims to test the provocative question PQ1. For tumors that arise from a pre-malignant field, what properties of cells in this field can be used to design strategies to inhibit the development of future tumors Recent studies showed that tumors are more than insular masses of proliferating cancer cells. Instead, they are complex tissues composed of multiple distinct cell types that participate in heterotypic interactions with one another. Here, we hypothesize that normal bone marrow mesenchymal stromal cells (MSCs) adjacent to the early premalignant MGUS cells are active participants in tumorigenesis and clonal evolution rather than passive bystanders; as such, these cells contribute to the development of multiple myeloma (MM). We will test this hypothesis in 3 specific Aims. In Specific Aim 1, we will elucidate genomic/transcriptomic events that govern alterations in MSCs at the premalignant stage of MGUS. We will perform integrative characterization of genomic, epigenomic and transcriptomic changes that occur in MSCs present near MGUS cells in bulk and at the single cell level. Further validation of specific target will be performed at the protein level to identify the spatial localization of these altered MSCs i the bone marrow niche using CyTOF mass spectrometry imaging. In Specific Aim 2, we will identify the sequential acquisition of genomic lesions in MSCs in the early premalignant stage and during disease progression in a murine model. Here, we will define the changes that occur in MSCs during the first genetic event in the premalignant plasma cells and identify changes that occur in the proximity of clonal plasma cells at the early MGUS stages vs. those that are present in distant bone marrow sites. We will examine whether MSCs alterations precede the early genetic hit (permissive microenvironment) or are acquired after MGUS development (acquired alterations). We will also define genomic and gene expression changes that occur in MSCs with stem cell aging in these mice. In Specific Aim 3, we will validate specific targets using CRISPR-based gene knockout of highly prioritized genes based on the studies performed in Aims 1 and 2 to functionally interrogate their specific role on MSCs and their regulation of tumor progression using in vitro and in vivo models. Furthermore, we will develop MSC-specific genomic alterations using CRISPR-cas9 mice crossed with Osx-cre mice to define the role of these target genes in regulating tumor initiation and MGUS progression in murine MM models. These focused research studies will help define the contributing role of MSCs in the early stages of MGUS development and clonal progression to MM. By identifying novel targets that regulate clonal evolution at the early premalignant stage of MGUS, we may be able to develop therapeutic agents that prevent or delay progression from MGUS to overt MM. Indeed, by eradicating the disease at the precursor stages, MM may become a preventable disease.